In recent years, progress has been made in dissecting the molecular events of prion protein (PrP) biogenesis. A novel topogenic sequence, termed the Stop Transfer Effector (STE), which directs nascent PrP in cell-free systems to either a doubly transmembrane or a secretory topology, has been identified. The choice between these topologic fates was shown to depend on the presence of a cytosolic factor. An intermediate with features of both topologic forms has been identified in vivo. Pathways by which alternate topologic fates and rapid ER degradation may occur have been identified. However, the relationship of these events to scrapie remains unknown. We propose to explore the role of this novel topogenic sequence in scrapie pathogenesis. The STE sequence will be mutagenized and the effects of mutations on PrP biogenesis and scrapie pathogenesis investigated. Mutants which alter steps in PrP biogenesis will be selected by cell-free transcription- linked translation and Xenopus oocyte microinjection. Some of these will be used to identify receptors and molecular chaperones with which nascent PrP interacts. Selected mutants will be studied in transgenic mice in order to determine if a relationship exists between unusual events in prion biogenesis and the pathogenesis of scrapie. Finally mutants transfected into N2a cells will be used to probe the effect of molecular chaperones on PrP biogenesis and parameters of scrapie infection.